The value of nanoparticulate compositions composed of perfluorocarbon nanoparticles coated with a surfactant layer to facilitate binding of desired components for imaging of various types is well established. See, for example, U.S. Pat. Nos. 5,690,907; 5,780,010; 5,989,520; 5,958,371; and PCT publication WO 02/060524, the contents of which are incorporated herein by reference. These documents describe emulsions of perfluorocarbon nanoparticles that are coupled to various targeting agents and to desired components, such as MRI imaging agents, radionuclides, and/or bioactive agents. Other compositions that have been used for targeted imaging include those disclosed in PCT publications WO 99/58162; WO 00/35488; WO 00/35887; and WO 00/35492. The contents of these publications are also incorporated herein by reference.
The integrin αvβ3, which binds to vitronectin, is recognized as a marker for neovasculature. It is relatively selective for activated endothelial cells and essentially unexpressed on mature, quiescent cells. Based on this characteristic, it has been attempted to use antagonists to this integrin as anticancer agents. Kerr, J. S., et al., Anticancer Res. (1999) 19:959-968 describe peptide mimetics which were able to decrease neovasculature formation in a mouse model system. U.S. Pat. No. 6,153,628 describes 1,3,4-thiadiazoles and 1,3,4-oxadiazoles that are αvβ3 antagonists and are said to useful in the treatment of disorders related to angiogenesis, including inflammation, bone degradation, tumors, metastases, thrombosis, and cell aggregation related conditions. U.S. Pat. Nos. 6,130,231 and 6,322,770 disclose fused heterocycles that are αvβ3 antagonists and useful for the same purposes, as does PCT publication WO 01/97848.
The WO 01/97848 publication discloses specific compounds that can be linked to ancillary substances, optionally through linker moieties, wherein these ancillary substances may include radionuclides, substances useful in magnetic resonance imaging, and X-ray contrast agents. This publication also discloses the use of these compounds coupled to certain ultrasound contrast agents, typically containing gaseous bubbles.
In addition to its expression in activated endothelial cells, αvβ3 is expressed on vascular smooth muscle cells, including macrophage in the walls of the vasculature. This complex binds cells to the surrounding matrix and is thus employed by cells in the course of migration. Accordingly, αvβ3 plays a role in restenosis by assisting the movement of cells into the lumen. A key component of restenosis involves vascular smooth muscle cell activation, proliferation and migration. Integrin heterodimers, in particular the αvβ3 integrin, are recognized as critical elements in these processes by providing cell adhesion to the extracellular matrix, inducing extracellular metalloproteinase expression, and facilitating smooth muscle cell migration. The αvβ3 integrin is widely distributed among endothelial cells, stimulated monocytes, T-lymphocytes, fibroblasts, vascular smooth muscle cells and platelets and binds to several extracellular matrix protein ligands including osteopontin, vitronectin, thrombospondin, and denatured collagens.
Antagonism of integrin mediated cell-matrix interactions within the balloon-stretched vessel walls inhibits inflammatory cell recruitment to the injury site, limits smooth muscle cell proliferation and migration, and diminishes extracellular matrix protein synthesis. Selective and nonselective blockade of integrins with cyclic RGD peptide antagonists have limited neointimal hyperplasia in several animal models of restenosis.
Restenosis is associated most often with angioplasty wherein, in an attempt to expand the vasculature using balloon catheters, the vasculature is broken, exposing the vascular smooth muscle cells. The resulting fractures require the movement of cells into the lumen; the αvβ3 acts to assist the migration through the matrix of collagen and fibrin to accomplish this. Accordingly, compositions that target αvβ3 may also be used to target smooth muscle cells and to image restenoses, in particular those associated with balloon angioplasty, and to deliver anti-proliferation agents such as paclitaxel, rapamycin, and other therapeutic moieties such as radionuclides, small molecules, peptides and nucleic acids.
While stent-based delivery systems offer the possibility of focal therapeutic drug effects within the tunica media of arteries without incurring the adverse side effects of systemic drug administration, and produce high local intimal concentrations of drug proximate to the stent-strut-arterial wall contact points, persistent high antiproliferative drug concentrations within the intima can impair arterial wall healing and reendothelialization, which promotes inflammation of the lumen lining and restenosis. The invention compositions avoid these problems.
It appears most peptidomimetics and neutralizing antibody αvβ3 antagonists have short half-lives and occupy the receptor for αvβ3 only transiently. The integrin-specific nanoparticles of the invention can target and block the binding of integrins exposed on smooth muscle cells by arterial overstretch injury as well as deliver a variety of therapeutic agents directly to cells that could inhibit inflammatory and restenosis processes and provide for molecular imaging for new, prognostic data relating the extent and severity of balloon injury to subsequent restenosis. The invention compositions avoid these problems.
Antibodies that are specific for αvβ3 integrin have been described in U.S. Pat. No. 6,171,588. These antibodies have been used in targeted magnetic resonance imaging (MRI) in a report by Sipkins, D. A., et al., Nature Med. (1998) 4:623-626; in this case coupled to the surface of liposomes via avidin linker proteins.
The use of antibodies directed to αvβ3 as a targeting agent for MRI using perfluorocarbon emulsions carrying chelated gadolinium has also been described by Anderson, S. A., et al., Magn. Reson. Med. (2000) 44:433-439, and in the above noted PCT publication WO 02/060524. Peptide ligands that are targeted to integrins have also been used as antagonists and have been suggested as a therapeutic strategy for rheumatoid arthritis by Storgard, C. M., et al., J. Clin. Invest. (1999) 103:47-53, who employed cyclic peptides containing the “RGD” type sequence known to interact with integrins.
Similar cyclic peptides were employed by Haubner, R., et al., J. Nucl. Med. (1999) 40:1061-1071 for tumor imaging by coupling the cyclic peptides directly to radionuclides. In an additional paper, the use of glycosylated forms of the cyclic peptides both for radiolabeling and PET is suggested by Haubner, R., et al., J. Nucl. Med. (2001) 42:326-336.
To applicants' knowledge, αvβ3-specific moieties other than antibodies have not been suggested for use as targeting agents in delivering image-aiding nanoparticulate emulsions or in delivering emulsions containing bioactive agents to regions containing activated endothelial cells such as sites of inflammation, tumors, atherosclerotic plaques, and restenoses.